This is a Phase II proposal to advance development of disubstituted tetrahydro-triazolo-pyrimidines (TTPs) as specific inhibitors of hepatitis B virus surface antigen (HBsAg) secretion in chronic infection. High serum levels of HBsAg (antigenemia) are believed to underlie immunotolerance toward the infection. Although they are clinically beneficial, the current small-molecule therapies rarely result in significant loss of serum HBsAg, elicit varying degrees of resistance, and are not curative. The parent compound of the TTPs was discovered in a high throughput screening campaign for HBsAg secretion inhibitors in a cell culture model. As proposed in our Phase I project, we 1) carried out extensive SAR studies to explore the salient features of the original structure, resulting in an increase in potency of more than 10-fold and improved chemical tractability; 2) confirmed the prediction that this compound family is active in inhibiting HBsAg secretion of HBV variants that are resistant to current small molecule drugs; and 3) identified formulation conditions for two lead compounds, improved solubility through salt formation, and characterized them in vivo for single dose ADMET properties. In Phase II of our project, we propose the following aims: 1) characterize the racemic properties of one lead compound, to determine whether enantiomeric separation will be necessary; 2) develop scale-up chemistry protocols amenable to eventual GMP manufacture, and produce sufficient non-GMP material for Phase II in vivo studies; 3) carry out dose range-finding ADMET studies on the lead compounds; 4) determine efficacy of the lead compounds on antigenemia in mouse and woodchuck models of hepatitis B, with an eye on possible toxicity due to HBsAg retention in the liver; 5) assess the potential for synergy in combination studies with our compound and current antivirals in cell culture and in vivo models; 6) submit the best lead compound to GLP- compliant IND-enabling studies. Concurrent with the above aims, we will 7) attempt to identify the precise molecular target of the TTPs through affinity studies. At the end of Phase 2, a TTP compound will be ready for IND filing and eventual clinical trials.